Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between - and ?-tubulin complexes, regulatory proteins, and the centrosomal matrix. Here we focus on determining the molecular mechanism of microtubule (MT) nucleation, and understanding the assembly and regulation of the nucleating machinery. Our laboratory is uniquely poised to use a hierarchy of structural approaches (x-ray crystallography, cryoEM single particle reconstruction, cryoEM Tomography) to determine the structures of ?-tubulin complexes in vitro and in situ, and to understand their mechanism of action through functional studies and innovative kinetic modeling. Previously we discovered that yeast ?-tubulin small complex (?TuSC ) can assemble into rings and obtained a 6.5A cryoEM structure of the rings, explaining the origins of MT 13-fold symmetry and discovering an unexpected mode of regulation and assembly. The proposed experiments expand upon these results, providing a detailed understanding of the physical origins of MT nucleation, and the cellular machinery that dictates it. Specifically we address the following questions: 1) Determine the structure of yeast and Drosophila ring complexes and their interactions with MTs: We will improve the resolution of our yeast ?TuSC rings and will generate a complete pseudo-atomic structure. We will extend the resolution (goal ~ 1nm) of our preliminary of cryoEM structure (3.5nm) of isolated Drosophila ?TuRCs (2.2MDa ?-tubulin ring complex) and identify how the different GCPs assemble around the ring. Structures of ?TuSC rings ?TuRCs bound to MTs or 1 layer of non-polymerizing yeast -tubulin will be determined and compared to structures of in situ capped MT minus ends from cryoEM tomography. 2) Mechanism of Spc110/72 facilitated assembly of ?TuSC rings and regulation by phosphorylation: We know that Spc110 stabilizes formation of yeast ?TuSC rings. We will use a newly developed FRET assay to efficiently measure ring assembly in vitro and determine what domains of Spc110 and Spc72 are required for assembly and the role of Spc110/72 phosphorylation. 3) Activation of nucleation by yeast ?TuSC rings: While necessary, assembly into rings is insufficient for potent MT nucleation, with a need for both ?TuSC closure to match MT symmetry and an allosteric activation. The role of PTMs or other binding partners in this process will be determined. 4) Structural organization of centrosomes - role of non ?-tubulin components: Our cryoEM tomography of basal bodies revealed new non-tubulin structures decorating the triplets. We propose a combination of SIM and STORM microscopy and cryoTomography to determine the molecular identity of these novel structures. Our SIM/STORM imaging has revealed unexpected structural domains within the centrosomal pericentriolar material. We continue these efforts assessing interactions and assembly including a structural analysis of Plp.